The objective of this proposal is to characterize the receptors and signaling pathways that mediate initial and sustained MLCzo phosphorylation and contraction in smooth muscle of the gut by excitatory neurotransmitters (acetylcholine and opioid peptides), and by modulatory endocannabinoids. Our recent studies have shown that contraction induced by Gq/n-coupled receptors (m3,motilin, CCK.A, S1P2,E!A) consists of a transient Ca2+-dependent phase mediated by MLC kinase, and a sustained, Ca2+-independent phase mediated by inhibition of MLC phosphatase (MLCP). Inhibition of MLCP is mediated by a RhoA- dependent pathway involving coordinated phosphorylation of two regulatory proteins, MYPT1 and CPI-17 that inhibit MLCP and induce sustained MLC20 phosphorylation and contraction. Gj-coupled receptors (e.g., 8-opioid), however, activated PI 3-kinase but not RhoA, implying operation of a distinct mechanism for inhibition of MLCP and sustained phosphorylation of MLCzo and contraction. Our preliminary studies indicate that the pathway involves PI 3-kinase-dependent activation of integrin-linked kinase (ILK). -Cannabinoid CBi receptors were coupled to an atypical G protein (Gc^/Gps/Cy-like RGS6) that precluded activation of downstream Gpy-dependent pathways. Accordingly, the specific aims of the proposalare: (1) to characterize 6-opioid, m2, and CBi receptor phosphorylation, desensitization, and internalization, and the roles of PKC-dependent RKIP (Raf-kinase inhibitory protein), Src-dependent caveolin-1, and "GRK2/GRK5-dependent clathrin in these processes;(2) to characterize regulation and cross-regulation at G protein level by specific RGS proteins (RGS12 for m2 and 8-opioid receptor-activated Ga,3 and Go^;RGS6 for CBj-activated Go^;and RGS4 and RGS16 for m3-activated Gctq and Gctn);and (3) to characterize novel downstream pathways for inhibition of MLC phosphatase and phosphorylation of MLCao by Gj-coupled receptors involving activation of ILK by PI 3-kinase, and the mechanisms for inactivation of ILK and CPI-7 via p38 MAP kinase-dependent PP2A and PP2C. Each specific aim is supported by substantial preliminary studies. These studies will provide a comprehensive analysis of novel signaling pathways initiated by prototypical Gj-coupled receptors in smooth muscle of the gut,and the interplay with signaling pathways initiated by Gq/n-coupled receptors. The studies will extend understanding of the function of Gj-coupled receptors in smooth muscle of the gut and in other tissues.